Pancreatic Cancer Cell Lines AsPC-1 Research Articles

Evaluation of Helium Ion Radiotherapy in Combination with Gemcitabine in Pancreatic Cancer In Vitro.

Pancreatic cancer is one of the most aggressive and lethal cancers. New treatment strategies are highly warranted. Particle radiotherapy could offer a way to overcome the radioresistant nature of pancreatic cancer because of its biological and physical characteristics. Within particles, helium ions represent an attractive therapy option to achieve the highest possible conformity while at the same time protecting the surrounding normal tissue. The aim of this study was to evaluate the cytotoxic efficacy of helium ion irradiation in pancreatic cancer in vitro. Human pancreatic cancer cell lines AsPC-1, BxPC-3 and Panc-1 were irradiated with photons and helium ions at various doses and treated with gemcitabine. Photon irradiation was performed with a biological cabin X-ray irradiator, and helium ion irradiation was performed with a spread-out Bragg peak using the raster scanning technique at the Heidelberg Ion Beam Therapy Center (HIT). The cytotoxic effect on pancreatic cancer cells was measured with clonogenic survival. The survival curves were compared to the predicted curves that were calculated via the modified microdosimetric kinetic model (mMKM). The experimental relative biological effectiveness (RBE) of helium ion irradiation ranged from 1.0 to 1.7. The predicted survival curves obtained via mMKM calculations matched the experimental survival curves. Mainly additive cytotoxic effects were observed for the cell lines AsPC-1, BxPC-3 and Panc-1. Our results demonstrate the cytotoxic efficacy of helium ion radiotherapy in pancreatic cancer in vitro as well as the capability of mMKM calculation and its value for biological plan optimization in helium ion therapy for pancreatic cancer. A combined treatment of helium irradiation and chemotherapy with gemcitabine leads to mainly additive cytotoxic effects in pancreatic cancer cell lines. The data generated in this study may serve as the radiobiological basis for future experimental and clinical works using helium ion radiotherapy in pancreatic cancer treatment.

MSLN induced EMT, cancer stem cell traits and chemotherapy resistance of pancreatic cancer cells

Chemoresistance is one of the main reasons for poor prognosis of pancreatic cancer. The effects of mesothelin (MSLN) on chemoresistance in pancreatic cancer are still unclear. We aim to investigate potential roles of MSLN in chemoresistance and its relationship with proliferation, epithelial-mesenchymal transition (EMT) and cancer stemness of pancreatic cancer cells. Human pancreatic cancer cell lines ASPC-1 and Mia PaCa-2 with high and low expression of MSLN, respectively, were selected. The ASPC-1 with MSLN knockout (KO) and Mia PaCa-2 of MSLN overexpression (OE) were generated. The effects of MSLN on cell phenotypes, expression of EMT-related markers, clone formation, tumor sphere formation, and pathologic role of MSLN in tumorigenesis were detected. Sensitivity of tumor cells to gemcitabine was evaluated. The results showed that adhesion, proliferation, migration and invasion were decreased significantly in ASPC-1 with MSLN KO, whereas increased significantly in Mia PaCa-2 with MSLN OE. The size and the number of clones and tumor spheres were decreased in ASPC-1 with MSLN KO, and increased in Mia PaCa-2 with MSLN OE. In xenograft model, tumor volume was decreased (tumor grew slower) in MSLN KO group compared to control group, while increased in MSLN OE group. Mia PaCa-2 with MSLN OE had a higher IC50 of gemcitabine, while ASPC-1 with MSLN KO had a lower IC50. We concluded that MSLN could induce chemoresistance by enhancing migration, invasion, EMT and cancer stem cell traits of pancreatic cancer cells. Targeting MSLN could represent a promising therapeutic strategy for reversing EMT and chemoresistance in pancreatic cancer cells.

APR-246 enhances the anticancer effect of doxorubicin against p53-mutant AsPC-1 pancreatic cancer cells

Pancreatic cancer (PC) is a disease with a high mortality rate, and the early diagnosis of PC is still challenging. The 5-year relative survival rate remains below 8%, and therapeutic strategies are ineffective at increasing patient survival rates. In PC cells, resistance to treatment has been associated with genetic changes that initiate aberrant developmental signaling pathways; therefore, new strategies for treating PC are warranted. Here, we sought to explore the small molecule p53 reactivator, APR-246, either as a mono-drug or combined with doxorubicin (Doxo) in the p53 mutant (p53-mut) AsPC-1 PC cell line. Cytotoxicity of APR-246 and Doxo was studied alone or in combination with the AsPC-1 cell line with MTT assay. CalcuSyn software was used to calculate the combination index (CI), which was based on the Chou and Talalay technique. Apoptosis was assessed after staining with Acridine orange/ethidium bromide (AO/EB). Morphological alterations were examined under a 200x magnification by an inverted microscope. Gene expression was quantified using quantitative reverse transcription PCR assay (RT-qPCR). APR-246 alone exhibited modest anti-proliferative effects, while Doxo combined with APR-246 showed synergistic effects associated with morphological change; no more enhanced apoptosis was observed, as supported by NOXA gene levels. In conclusion, APR-246, whether monotherapy or combined with Doxo, was hindering the proliferation of p53-mut AsPC-1 PC cells, and further in vitro and in vivo research is warranted.

Abstract 4500: Enedione derivates as a potential cancer treatment through the inhibition of COPZ1

Abstract Cancer is a leading cause of death worldwide; it was responsible for approximately 10 million deaths in 2020. Besides surgery for tumor removal, the most common treatments available are chemotherapy and radiation, which are not selective, resulting in side effects such as pain, hair loss, nausea, and vomiting. They are also ineffective against stem-like and dormant tumor cells, which could lead to a relapse and/or metastasis. Consequently, the development of drugs with tumor-specific targets is needed. COPI is a coatomer responsible for the retrograde transport from Golgi complex (GC) to the endoplasmic reticulum (ER). In this context, we developed four compounds targeting disruption of GC through inhibition of COPI and previous results showed that compounds 1-4 significantly decrease cell viability of pancreatic cancer cell lines MIA PaCa-2 and AsPC-1, compared to hTERT-HPNE normal pancreatic cell line. In the present work, RT q-PCR confirmed that the two isoforms of the COPI ζ subunit COPZ1 and COPZ2 are expressed at similar levels in normal cells while COPZ2 is downregulated in tumor cell lines. Our in vitro results demonstrated that compounds 1-4 decreased cell viability of breast cancer (HCC1143) and melanoma (A-395) cell lines. The tested compounds are able to target COPZ1-dependent tumor cells, therefore they are great candidates for further studies aiming to develop a selective treatment for cancer. Citation Format: Allana C. Martins, Barbara Mitsuyasu Barbosa, Ingridhy O. Maia Freitas da Silveira, Roberto Gomes. Enedione derivates as a potential cancer treatment through the inhibition of COPZ1. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4500.

Treatment of Human Pancreatic Cancers Following Local and Systemic Administration of Oncolytic Adenovirus Serotype 35.

Oncolytic adenoviruses (Ads) (OAds) are gaining attention as an effective remedy for pancreatic cancer. Most OAds are based on human Ad serotype 5 (Ad5) (OAd5); however, two major drawbacks of OAd5 have been reported. Expression of coxsackievirus-adenovirus receptor, a primary infection receptor of Ad5, is often decreased on malignant tumor cells, including pancreatic cancers. More than 60% of adults have neutralizing antibodies against Ad5. Previously, we developed an OAd composed of Ad serotype 35 (Ad35) (OAd35). Ad35 recognizes CD46, which is often up-regulated on pancreatic cancers. In addition, only 20% or fewer adults have anti-Ad35 neutralizing antibodies. We examined the tumor cell lysis activities of OAd35 in the four human pancreatic cancer cell lines in the presence and absence of human serum. The tumor growth suppression effects of OAd35 after local and systemic administration were evaluated in nude mice bearing human pancreatic tumors. OAd35 showed higher levels of tumor cell lysis activities than OAd5 in the human pancreatic cancer cell lines AsPC-1 and BxPC-3. Although the in vitro tumor cell lysis activities of OAd5 against MIA PaCa-2 and PANC-1 cells were strongly attenuated in the presence of human serum, OAd35 mediated comparable levels of tumor cell lysis in the presence and absence of human serum. Systemic administration of OAd5 did not mediate significant growth inhibition against the subcutaneous BxPC-3 tumor. On the other hand, OAd35 significantly suppressed tumor growth. OAd35 would be suitable as an alternative anticancer agent for pancreatic cancer.

Gallic acid markedly stimulates GLUT1-mediated glucose uptake by the AsPC-1 pancreatic cancer cell line.

Phenolic acids are recognized as chemopreventive and chemotherapeutic agents. Altered glucose and glutamine metabolism are recognized hallmarks of cancer cells. We aimed to test the influence of phenolic acids on glucose and glutamine cellular uptake by a breast (MCF-7) and a pancreatic (AsPC-1) cancer cell line. Several phenolic acids (caffeic, ferrulic, proctocatechuic, coumaric and gallic acid) affected 3H-glutamine and/or 3H-deoxy-d-glucose (3H-DG) uptake. Gallic acid (100µM) caused a 3-fold increase in 3H-DG uptake by AsPC-1 cells, associated with a 3.7-fold increase in lactic acid production. Gallic acid stimulated GLUT1-mediated 3H-DG uptake and increased the affinity of this transporter for 3H-DG. We further verified that gallic acid does not change GLUT1 transcription rates and cellular redox state and that its effect does not involve PI3K, mTOR and MAP kinases and is not associated with a proproliferative effect. Gallic acid also increased 3H-DG uptake by MCF-7 cells, although less potently. Further investigation is necessary to elucidate the cellular pathways involved in this effect of gallic acid.

AGE/RAGE axis regulates reversible transition to quiescent states of ALK-rearranged NSCLC and pancreatic cancer cells in monolayer cultures

Cancer recurrence due to tumor cell quiescence after therapy and long-term remission is associated with cancer-related death. Previous studies have used cell models that are unable to return to a proliferative state; thus, the transition between quiescent and proliferative states is not well understood. Here, we report monolayer cancer cell models wherein the human non-small cell lung carcinoma cell line H2228 and pancreatic cancer cell line AsPC-1 can be reversibly induced to a quiescent state under hypoxic and serum-starved (HSS) conditions. Transcriptome and metabolome dual-omics profiles of these cells were compared with those of the human lung adenocarcinoma cell line A549, which was unable to enter a quiescent state under HSS conditions. The quiescence-inducible cells had substantially lower intracellular pyruvate and ATP levels in the quiescent state than in the proliferative state, and their response to sudden demand for energy was dramatically reduced. Furthermore, in quiescence-inducible cells, the transition between quiescent and proliferative states of these cells was regulated by the balance between the proliferation-promoting Ras and Rap1 signaling and the suppressive AGE/RAGE signaling. These cell models elucidate the transition between quiescent and proliferative states, allowing the development of drug-screening systems for quiescent tumor cells.

The effect of alloferon on chemosensitivity of pancreatic cancer through the regulation of SLC6A14 expression

Abstract Pancreatic cancer is one of the most malignant solid tumors with a high mortality rate. Although there have been many trials for effective therapies using chemotherapeutic drugs, such as gemcitabine, pancreatic cancer still shows significantly higher mortality than other types of cancer. Therefore, it is needed to examine the effective therapeutic ways to improve conventional therapy for pancreatic cancer. It has been previously reported that cancer cells possess a high rate of glutamine uptake needed for their rapid proliferation. In addition, it has recently been reported that amino acid transporter SLC6A14 is up-regulated in some cancers in close relation to glutamine metabolism. Alloferon is a peptide isolated from the insect immune system and shows anti-viral and anti-inflammatory effects based on its immune modulating function. In addition, its anti-tumor effect was recently discovered, but the related mechanisms are largely unknown. For this reason, I investigated the effect of alloferon on pancreatic cancer cell lines Panc-1 and AsPC-1. When Panc-1 and AsPC-1 were exposed to alloferon for 3 weeks, I found down-regulation of SLC6A14 expression and decreased glutamine uptake. Given that SLC6A14 is involved in tumor progression and survival by promoting glutamine uptake into cancer cells, alloferon could be an effective way with conventional chemotherapeutic drug, gemcitabine. Therefore, alloferon could be a useful adjuvant for an effective therapeutic way with conventional chemotherapeutic drug, gemcitabine. This study was funded by the NRF of Korea (No.:2017R1A2B2010948, 2017R1A6A3A11032576, 2020R1C1C1009334)

The Nitrated Form of Nateglinide Induces Apoptosis in Human Pancreatic Cancer Cells Through a Caspase-dependent Mechanism.

Nitric oxide (NO) has antitumor activity against various solid tumor cell types in addition to its vasodilatory effect. In the current study, we investigated the effect and mechanism of the synthetic nitrated form (NO2-NAT) of nateglinide, a hypoglycemic agent, on the induction of cell death in human pancreatic cancer cells. NO production was evaluated by measuring nitrite (NO2) and nitrate (NO3) (NOx). Apoptotic cell numbers were determined using annexin V. NO2-NAT released nitrate and nitrite ions immediately upon dissolving in aqueous solution. NO2-NAT caused significant extracellular leakage of lactate dehydrogenase (LDH) in the human pancreatic cancer cell lines AsPC1 and BxPC3, and increased annexin-positive cells in a time- and concentration-dependent manner. NO2-NAT also significantly increased the activity of caspases 3 and 7. Exposure to Z-VAD-FMK, a caspase inhibitor, significantly suppressed NO2-NAT-induced cell death. These results indicated that NO2-NAT induces apoptosis in human pancreatic cancer cells and may serve as a new NO-based chemotherapeutic agent for the treatment of pancreatic cancer.

Abstract PO-092: Influence of the IL-13-receptor alpha 1 chain on the malignant phenotype of pancreatic cancer cells

Abstract Introduction: Increasing evidence indicates salient activities of Interleukin (IL)-4, IL-13 and their specific Type-II-IL-4 receptor complex (IL-4Rα/IL-13Rα1) in carcinomas including pancreatic cancer. While IL-4 seems to be tumor-promoting in PDAC, the specific role of the IL-13/IL-13Rα1 axis was yet to be determined. Both cytokines are abundant in PDAC through cells of the tumor microenvironment. In this project, we planned to determine the specific effects of the IL-13Rα1-receptor chain on the malignant phenotype of pancreatic cancer cells. Methods: Expression of IL-13Rα1, IL-4Rα, and the human common gamma chain (γc) in cultured pancreatic cancer cell lines AsPC-1, Capan-1, PANC-1, MIA PaCa-2 and A818-6 as well as downstream signal transduction factors of IL13-Rα1 were detected by Western blot. The role of IL-13Rα1 in pancreatic cancer cell proliferation, mobility and migration was demonstrated based on the successful establishment of stable shRNA based IL-13Rα1-knockdown (KD) clones in Capan-1 and MIA PaCa-2 cell lines. Results: Not only the basal anchorage-dependent growth but also the anchorage-independent growth in soft agar were inhibited in Capan-1 and MIA PaCa-2 cells due to IL-13Rα1-downregulation. Mechanistically, IL-13Rα1-KD was associated with increased apoptosis. Additionally, signal transduction factors STAT3, STAT6, PI3K, Akt and ERK1/2 were activated in Capan-1 exposed to exogenous IL-4 and IL-13. Interestingly, IL-13Rα1-KD reduced the basal expression of STAT3, Akt and ERK1/2. IL-4 and IL-13 induced activation of STAT3, ERK1/2, Akt and STAT6 was significantly suppressed in Capan-1-IL-13Rα1-KD (C-KD) clones. Furthermore, IL-13Rα1-KD resulted in enhanced mobility and migration in Capan-1 cells. Conclusion: It was demonstrated that endogenous IL-13Rα1 is vital for pancreatic cancer cell growth with the involvement of STAT3/6, ERK1/2 and Akt. Surprisingly, IL-13Rα1-KD resulted in enhanced mobility and migration. Overall, IL-13Rα1 plays a critical but heterogenous role in PDAC. Our findings increase the understanding of the different functions and mechanisms involving IL-13Rα1 in pancreatic cancer progression. Citation Format: Jingwei Shi, Marko Kornmann, Benno Traub. Influence of the IL-13-receptor alpha 1 chain on the malignant phenotype of pancreatic cancer cells [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2021 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2021;81(22 Suppl):Abstract nr PO-092.

Effects of Metallic and Carbon-Based Nanomaterials on Human Pancreatic Cancer Cell Lines AsPC-1 and BxPC-3.

Pancreatic cancer, due to its asymptomatic development and drug-resistance, is difficult to cure. As many metallic and carbon-based nanomaterials have shown anticancer properties, we decided to investigate their potential use as anticancer agents against human pancreatic adenocarcinoma. The objective of the study was to evaluate the toxic properties of the following nanomaterials: silver (Ag), gold (Au), platinum (Pt), graphene oxide (GO), diamond (ND), and fullerenol (C60(OH)40) against the cell lines BxPC-3, AsPC-1, HFFF-2, and HS-5. The potential cytotoxic properties were evaluated by the assessment of the cell morphology, cell viability, and cell membrane damage. The cancer cell responses to GO and ND were analysed by determination of changes in the levels of 40 different pro-inflammatory proteins. Our studies revealed that the highest cytotoxicity was obtained after the ND treatment. Moreover, BxPC-3 cells were more sensitive to ND than AsPC-1 cells due to the ND-induced ROS production. Furthermore, in both of the cancer cell lines, ND caused an increased level of IL-8 and a decreased level of TIMP-2, whereas GO caused only decreased levels of TIMP-2 and ICAM-1 proteins. This work provides important data on the toxicity of various nanoparticles against pancreatic adenocarcinoma cell lines.

Development and Evaluation of Icariin-Loaded PLGA-PEG Nanoparticles for Potentiation the Proapoptotic Activity in Pancreatic Cancer Cells.

Therapeutic efficacy of antineoplastic agents possessing a selective target to the nucleus of the cancer cells could be enhanced through novel formulation approaches. Thus, towards improvement of anticancer potential of icariin (ICA) on pancreatic cancer, the drug was entrapped into the polymeric poly lactic-co-glycolic acid (PLGA) with polyethylene glycol (PEG) as diblock copolymer. Optimization of the formulation was done using Statgraphics software to standardize percentages of PEG-PLGA and tween 80 (TW80) to obtain the smallest particle size. The optimized formulation was found to be in nanometer size and low PDI (0.321). Optimized formula enhanced cytotoxicity and apoptotic potential, compared with ICA-raw, against pancreatic cancer cell lines (aspc-1). The entrapment efficiency of the polymeric micelles was 72.34 ± 2.3% with 93.1 ± 6.5% release of ICA within 72h. There was a twofold increase in apoptosis and sevenfold increase in necrosis of aspc-1 cells when incubated with raw ICA compared to control cells. Further, loss of mitochondrial membrane potential (⁓50-fold) by the ICA-loaded PMs and free drug compared to control cells was found to be due to the generation of ROS. Findings of cell cycle analysis revealed the significant arrest of G2-M phase of aspc-1 cells when incubated with the optimized formulation. Simultaneously, a significantly increased number of cells in pre-G1 revealed maximum apoptotic potential of the drug when delivered via micellar formulation. Finally, upregulation of caspase-3 established the superiority of the PMs approach against pancreatic cancer. In summary, the acquired results highlighted the potentiality of PMs delivery tool for controlling the growth of pancreatic cancer cells for improved efficacy.

Comprehensive Analysis of E3 Ubiquitin Ligases Reveals Ring Finger Protein 223 as a Novel Oncogene Activated by KLF4 in Pancreatic Cancer.

Pancreatic cancer is one of the major malignancies and causes of mortality worldwide. E3 ubiquitin–protein ligases transfer activated ubiquitin from ubiquitin-conjugating enzymes to protein substrates and confer substrate specificity in cancer. In this study, we first downloaded data from The Cancer Genome Atlas pancreatic adenocarcinoma dataset, acquired all 27 differentially expressed genes (DEGs), and identified genomic alterations. Then, the prognostic significance of DEGs was analyzed, and eight DEGs (MECOM, CBLC, MARCHF4, RNF166, TRIM46, LONRF3, RNF39, and RNF223) and two clinical parameters (pathological N stage and T stage) exhibited prognostic significance. RNF223 showed independent significance as an unfavorable prognostic marker and was chosen for subsequent analysis. Next, the function of RNF223 in the pancreatic cancer cell lines ASPC-1 and PANC-1 was investigated, and RNF223 silencing promoted pancreatic cancer growth and migration. To explore the potential targets and pathways of RNF223 in pancreatic cancer, quantitative proteomics was applied to analyze differentially expressed proteins, and metabolism-related pathways were primarily enriched. Finally, the reason for the elevated expression of RNF223 was analyzed, and KLF4 was shown to contribute to the increased expression of RNF233. In conclusion, this study comprehensively analyzed the clinical significance of E3 ligases. Functional assays revealed that RNF223 promotes cancer by regulating cell metabolism. Finally, the elevated expression of RNF223 was attributed to KLF4-mediated transcriptional activation. This study broadens our knowledge regarding E3 ubiquitin ligases and signal transduction and provides novel markers and therapeutic targets in pancreatic cancer.

Quantitative Super-Resolution Imaging for the Analysis of GPCR Oligomerization.

G-protein coupled receptors (GPCRs) are known to form homo- and hetero- oligomers which are considered critical to modulate their function. However, studying the existence and functional implication of these complexes is not straightforward as controversial results are obtained depending on the method of analysis employed. Here, we use a quantitative single molecule super-resolution imaging technique named qPAINT to quantify complex formation within an example GPCR. qPAINT, based upon DNA-PAINT, takes advantage of the binding kinetics between fluorescently labelled DNA imager strands to complementary DNA docking strands coupled to protein targeting antibodies to quantify the protein copy number in nanoscale dimensions. We demonstrate qPAINT analysis via a novel pipeline to study the oligomerization of the purinergic receptor Y2 (P2Y2), a rhodopsin-like GPCR, highly expressed in the pancreatic cancer cell line AsPC-1, under control, agonistic and antagonistic conditions. Results reveal that whilst the density of P2Y2 receptors remained unchanged, antagonistic conditions displayed reduced percentage of oligomers, and smaller numbers of receptors in complexes. Yet, the oligomeric state of the receptors was not affected by agonist treatment, in line with previous reports. Understanding P2Y2 oligomerization under agonistic and antagonistic conditions will contribute to unravelling P2Y2 mechanistic action and therapeutic targeting.

MiR-373 Suppresses Cell Proliferation and Apoptosis via Regulation of SIRT1/PGC-1α/NRF2 Axis in Pancreatic Cancer.

Objective Our study aimed to investigate function and mechanism of miR-373 in proliferation and apoptosis of pancreatic cancer (PC) cells by regulating NAD+-dependent histone deacetylase sirtulin 1 (SIRT1). Materials and Methods This experimental study included two PC cell lines AsPC-1 and PANC-1 in which expression levels of miR-373 and SIRT1 were manipulated. The level of miR-373 was detected by reverse transcription quantitative polymerase chain reaction (RT-qPCR) method. Expression levels of SIRT1, BCL-2, BAX, cleaved CASPASE-8/9/3, PARP, PGC-1α, NRF2, eNOS and iNOS were examined via RT-qPCR and western blotting, respectively. The binding sites of miR-373 on the SIRT1 were examined via dual-luciferase assay. Cell proliferation and apoptosis were examined by MTT assay, colony formation assay, Annexin-V/PI staining and TUNEL assay. The oxidative metabolic changes were monitored by reactive oxygen species (ROS), malondialdehyde (MDA) and superoxide dismutase (SOD) detection. Results miR-373 could specifically target the 3’-UTR of SIRT1 and reduce its expression in PC cells. Either elevated expression of miR-373 or partial loss of SIRT1 inhibited cell proliferation and induced cell apoptosis. Accumulation of BAX and cleaved CASPASE-8/9/3, inhibition of PGC-1α/NRF2 pathway, increase oxidative stress and reduction of BCL-2 as well as uncleaved PARP were found in the presence of miR-373 or the absence of SIRT1. Overexpression of SIRT1 could reduce anti-proliferative and pro-apoptotic effects of miR-373. ConclusionOverall, this study concluded that miR-373-dependent SIRT1 inhibition displays anti-proliferative and pro- apoptotic roles in PC cells via PGC-1α/NRF2 pathway, which highlights miR-373 as a potential target for PC treatment.

Abstract PO-039: Inhibition of migration, invasion and drug resistance of pancreatic adenocarcinoma cells- Role of Snail, Slug and Twist and small molecule Inhibitors

Abstract Purpose: The main purpose of this study is to demonstrate the effects of epithelial to mesenchymal transition activating transcription factor silencing (EMT-ATF silencing) on migration, invasion, drug resistance and tumor forming abilities of various pancreatic cancer cell lines. Additionally, the contribution of small molecule inhibitors of EMT (SD-208 and CX4945) to the effects of gene silencing was evaluated. Methods: EMT activating transcription factors “Snail, Slug and Twist” were silenced by short hairpins on Panc-1, MIA PaCa-2, BxPC-3, and AsPC-1 pancreatic cancer cell lines. The changes in migration, invasion, laminin attachment, cancer stem-like cell properties and tumor forming abilities were investigated. Chemo sensitivity assays and small molecule inhibitors of EMT were applied to the metastatic pancreatic cancer cell line AsPC-1. Results: EMT-ATF silencing reduce EMT and stem cell-like characteristics of pancreatic cancer cell lines. Following EMT-ATF silencing amongst the four PC cell lines, AsPC-1 showed the best response and was chosen for further chemo resistance and combinational therapy applications. EMT downregulated AsPC-1 cells showed less resistance to selected chemotherapeutics compared to the control group. Both small molecule inhibitors enhanced the outcomes of EMT-ATF silencing. Conclusion: Overall it was found that EMT-ATF silencing, either by EMT-ATF silencing or with the enhancement by small molecules, is a good candidate to treat pancreatic cancer since it simultaneously minimizes metastasis, stem cell properties, and drug resistance. Citation Format: Ezgi Kasikci, Esra Aydemir, Omer Faruk Bayrak, Fikrettin Sahin. Inhibition of migration, invasion and drug resistance of pancreatic adenocarcinoma cells- Role of Snail, Slug and Twist and small molecule Inhibitors [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2020 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2020;80(22 Suppl):Abstract nr PO-039.

Anticancer effects of miR-124 delivered by BM-MSC derived exosomes on cell proliferation, epithelial mesenchymal transition, and chemotherapy sensitivity of pancreatic cancer cells.

Objective: This study aims to explore the roles of miR-124 in pancreatic tumor and potential vehicles.Results: The miR-124 expression levels decreased in pancreatic adenocarcinoma tissues and cancer cell lines AsPC-1, PANC1, BxPC-3 and SW1990. Furthermore, the elevated expression of miR-124 in AsPC-1 and PANC1 via miR-124 mimic transfection-induced apoptosis, metastasis and epithelial mesenchymal transition was suppressed, and the EZH2 overexpression partly reversed the protective effects of miR-124 against pancreatic tumors. In addition, the expression of miR-124 was detected in exosomes extracted from miR-124-transfected BM-MSCs, and these exosomes delivered miR-124 into pancreatic cancer cells, and presented the anti-tumor effects in vitro and in vivo.Conclusion: MiR-124-carried BM-MSC-derived exosomes have potential applications for the treatment of pancreatic tumors.Methods: The expression of miR-124 and EZH2 was determined in both pancreatic cancer tissues and cell lines. miR-124 or EZH2 was overexpressed in AsPC-1 and PANC1 cells. Then, the effects on cell viability. apoptosis, invasion, migration and epithelial mesenchymal transition were evaluated. Afterwards, the roles of miR-124 on the expression and function of EZH2 in pancreatic tumors were determined by dual luciferase reporter assay. Subsequently, miR-124 was transfected to bone marrow mesenchymal stromal cells (BM-MSCs), and the BM-MSCs derived exosomes were isolated and co-cultured with AsPC-1 and PANC1 cells, or injected into pancreatic cancer tumor-bearing mice.

Pancreatic circulating tumor cell detection by targeted single-cell next-generation sequencing.

Single-cell next-generation sequencing (scNGS) technology has been widely used in genomic profiling, which relies on whole-genome amplification (WGA). However, WGA introduces errors and is especially less accurate when applied to single nucleotide variant (SNV) analysis. Targeted scNGS for SNV without WGA has not been described. We aimed to develop a method to detect circulating tumor cells (CTCs) with DNA SNVs. We tested this targeted scNGS method with three driver mutant genes (KRAS/TP53/SMAD4) on one pancreatic cancer cell line AsPC-1 and then applied it to patients with metastatic PDAC for the validation. All single-cell of AsPC-1 and spiked-in AsPC-1cells in healthy donor blood, which were isolated by the filtration with size or by flow cytometry, were detected by targeted scNGS method. All blood samples from six patients with metastatic PDAC, for the validation of target scNGS method, showed CTCs with SNVs of KRAS/TP53/SMAD4 and the positive confirmation of immunofluorescent stainings with Pan-CK/Vimentin/CD45. Four patients with early stage disease, one patient with benign pancreatic cyst and a healthy control sample all showed concordant results between targeted scNGS and CTC enumeration. The novel technique of targeted scNGS for SNV analysis, without pre-amplification, is a promising method for identifying and characterizing circulating tumor cells.

To Study theAnti-cancer Effects of Shigella Flexneri in AspC-1 Pancreatic Cancer Cell Line in Approach to Bax and bcl-2 Genes.

Cancer is the uncontrolled division of cells and can be caused by genetic or environmental factors. Pancreatic cancer is one of the deadliest among all cancers. The role of bacteria as an anticancer agent dates back to almost 100years ago. The microbiome has recently become a focus of research in carcinogenesis and even pancreatic cancer. Shigella flexneri is a gram-negative bacterium, which causes shigellosis with symptoms such as diarrhea, fever, and stomach cramps in human. Shigella flexneri may play a very important role in the internal pathways of apoptosis and may induce apoptosis in some of the cancerous cells. In this experiment bacteria were cultured on Salmonella-Shigella agar, then inoculated into BHI Broth medium. After sonication, the protein concentration of the bacterium was measured by using the ZellBio Sensitive Protein Bradford Assay kit. MTT assay was performed to obtain IC50 for the said bacterial protein. Later by cDNA kit synthesized the cDNA based on the RNA template. In the end, the results were analyzed using real-time PCR and the expression of Bax and Bcl-2 genes was measured before and after treatment. The results showed that Shigella flexneri has the potential anti-proliferative effect in pancreatic cancer. The inhibitory concentration, pro-apoptotic amount to upregulate Bax, and meanwhile also to downregulate the bcl-2 found to be 10μl. In general, due to defects in the apoptotic pathway in cancer cells and the existence of drug-resistant cells, the detection of new apoptotic inducers such as Shigella flexneri cell extract can be used for further studies on cancer therapy.

Inhibition of Migration, Invasion and Drug Resistance of Pancreatic Adenocarcinoma Cells - Role of Snail, Slug and Twist and Small Molecule Inhibitors.

PurposeThe main purpose of this study is to demonstrate the effects of epithelial to mesenchymal transition activating transcription factor silencing (EMT-ATF silencing) on migration, invasion, drug resistance and tumor-forming abilities of various pancreatic cancer cell lines. Additionally, the contribution of small molecule inhibitors of EMT (SD-208 and CX4945) to the effects of gene silencing was evaluated.MethodsEMT activating transcription factors “Snail, Slug and Twist” were silenced by short hairpins on Panc-1, MIA PaCa-2, BxPC-3, and AsPC-1 pancreatic cancer cell lines. The changes in migration, invasion, laminin attachment, cancer stem-like cell properties and tumor-forming abilities were investigated. Chemosensitivity assays and small molecule inhibitors of EMT were applied to the metastatic pancreatic cancer cell line AsPC-1.ResultsEMT-ATF silencing reduced EMT and stem cell-like characteristics of pancreatic cancer cell lines. Following EMT-ATF silencing amongst the four PC cell lines, AsPC-1 showed the best response and was chosen for further chemoresistance and combinational therapy applications. EMT downregulated AsPC-1 cells showed less resistance to select chemotherapeutics compared to the control group. Both small molecule inhibitors enhanced the outcomes of EMT-ATF silencing.ConclusionOverall it was found that EMT-ATF silencing, either by EMT-ATF silencing or with the enhancement by small molecules, is a good candidate to treat pancreatic cancer since it simultaneously minimizes metastasis, stem cell properties, and drug resistance.